Five acriflavine agglutination test-positive (acf⁺) colonies and five negative (acf^-) colonies were isolated from each of the four strains (Ebina, CMB2, N9, and Schu) of Francisella tularensis, and the correlation between the virulence in experimental mice and the entry and intracellular multiplication in cultured mouse fibroblast cells (L-929 cells) was examined. All of the acf^- colonies derived from the Ebina and CMB2 strains were highly virulent in mice, readily entering and growing well in the cells, while all of the acf^- colonies from N9 and Schu strains were of low virulence and neither entered nor grew in the cells effectively. On the other hand, regardless of their parent strains, the acf⁺ colonies were low virulent and most of those colonies did neither enter nor grow in L-929 cells. In addition, two acf^- colonies, one from the N9 and the other from the Schu strain, gained virulence through several passages in mice, and in parallel, their entry and multiplication also improved. However, two acf⁺ colonies from the Ebina strain and one acf⁺ colony from the N9 strain showed a moderate degree of the entry and multiplication although they were all low virulent. The overall results indicate that the entry and multiplication in cells are important factors regulating the virulence of F. tularensis. The results also showed, however, that they were not sole factors to elucidate the virulence of the bacterium in mice.